Preparation and properties of T-ZnOw enhanced BCP scaffolds with double-layer structure by digital light processing

Bone scaffolds require both good bioactivity and mechanical properties to keep shape and promote bone repair. In this work, T-ZnOw enhanced biphasic calcium phosphate (BCP) scaffolds with triply periodic minimal surface (TPMS)-based double-layer porous structure were fabricated by digital light processing (DLP) with high precision. Property of suspension was first discussed to obtain better printing quality. After sintering, T-ZnOw reacts with β-tricalcium phosphate (β-TCP) to form Ca19Zn2(PO4)14, and inhibits the phase transition to α-TCP. With the content of T-ZnOw increasing from 0 to 2 wt%, the flexural strength increases from 40.9 to 68.5 MPa because the four-needle whiskers can disperse stress, and have the effect of pulling out as well as fracture toughening. However, excessive whiskers will reduce the cure depth, and cause more printing defects, thus reducing the mechanical strength. Besides, T-ZnOw accelerates the deposition of apatite, and the sample with 2 wt% T-ZnOw shows the fastest mineralization rate. The good biocompatibility has been proved by cell proliferation test. Results confirmed that doping T-ZnOw can improve the mechanical strength of BCP scaffolds, and keep good biological property, which provides a new strategy for better bone repair.

Ca/P ceramic is one of the most promising material for bone scaffold because of its similar element component to natural bone [6]. Hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 , HA) and -tricalcium phosphate (Ca 3 (PO 4 ) 2 , β-TCP) have been widely used in bone scaffold, and biphasic calcium phosphate (BCP), the mixture of them, has a suitable degradation rate and mechanical properties [7][8][9][10]. However, the bioactivity and strength of BCP ceramic still need to be enhanced. Some people tried to improve the bone repair ability and strength of ceramic by adding ions such as Fe [11,12], Ag [13,14], Zr [15], Zn, and so on. Zn is one of the essential trace elements in the human body. The total weight of Zn in human body is about 1.4-2.3 g, with about 50% deposit in bones [16,17]. Tetrapod-like zinc oxide whisker (T-ZnO w ) is the only one of whiskers that has a spatial structure: Four needles of it stretch to different directions, offering an isotropic performance and helping to improve mechanical properties [18][19][20][21]. At the same time, T-ZnO w shows excellent antibacterial properties and good biocompatibility [22,23], which is beneficial to scaffolds used for implanting. Therefore, the addition of T-ZnO w is hopeful to enhance the strength and bone repair ability of composite bioceramics.
Good scaffolds require not only suitable material but also porous structure to induce cells to grow in. Pores' size, distribution, shape, and connection condition all have great impact on the performance of scaffolds [24][25][26]. Triply periodic minimal surface (TPMS) is a commonly-used structure. The curvature of any point on the surface of TPMS structure is zero, so stress can be uniformly distributed to avoid the damage caused by stress concentration. By adjusting parameters, the pore size and repeating unit of TPMS can be adjusted to meet different requirements [27,28]. However, it is hard for traditional methods to fabricate this structure considering the complexity, and fortunately, additive manufacturing (AM) techniques, including selective laser sintering (SLS) [29,30], direct ink writing (DIW) [31], stereolithography (SL) [32], digital light processing (DLP) [33,34], and so on, provide a new way to solve this problem by printing layer by layer [35,36]. Compared with other AM methods, DLP is based on suspension and more suitable to fabricate complex ceramic scaffolds with high precision [37].
The rheological property and cure property of suspension have great influence on DLP printing, and need to be studied to get high precision. Li et al. [38] investigated the cure depth and excess cure width of the suspension with different kinds of ZrO 2 powders, and found that as the colorant content increases, the cure depth and excess cure width both decrease due to the ceramic absorbance. Zheng et al. [39] researched the size distribution of ternary Al 2 O 3 particles and their effect on the rheology of ceramic suspension. An optimized proportion formula was derived to get the suspension for DLP with high solid loading and low viscosity. T-ZnO w has different particle morphology and optical properties from BCP, but researches on BCP suspension containing T-ZnO w are still inadequate. It is necessary to investigate the viscosity and cure depth of suspension containing T-ZnO w to achieve the best printing quality.
In this work, T-ZnO w enhanced BCP scaffolds with TPMS-based double-layer porous structure were fabricated by DLP technology. The rheological and cure property of suspension was first discussed. Then phase composition, microstructure, shrinkage, volume density, and mechanical strength were tested to identify the influence of T-ZnO w . Finally, apatite-forming ability and cell proliferation were investigated to verify the biological property.

1 Raw material
The ceramic powders were Ca 10 (PO 4 ) 6 (OH) 2 (HA, Kunshan Chinese Technology New Material Co., Ltd., China, index  1.665), Ca 3 (PO 4 ) 2 (β-TCP, Kunshan Chinese Technology New Material Co., Ltd., China, index  1.626), and T-ZnO w (Chengdu Crystrealm Co., Ltd., China, index  2.008). As shown in Figs. 1(a)-1(c), the powder of HA has the minimum size which is at nanoscale, while the size of β-TCP is much larger. The size of T-ZnO w varied greatly with the length of needles ranging from 5 to 50 μm. The particle size distribution of BCP and T-ZnO w is shown in Fig. 1(d). 1,6-hexanediol diacrylate (HDDA, Chengdu Fourth City New Material Co., Ltd., China) and tripropylene glycol diacrylate (TPGDA, Chengdu Fourth City New Material Co., Ltd., China) were used as the UV resin monomers. To get well-performed ceramic slurry, Solsperse 41000 (Lubrizol, Spain) was used as the dispersant. Diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO, BASF, Germany) was chosen as the photoinitiator, and 4-methoxyphenol (MEHQ, Sinopharm Chemical Reagent Co., Ltd., China) was chosen as the photoinhibitor. Defoamer Foamex N (TEGO, Germany) was used to reduce the bubbles in suspension during printing. Simulated body fluid (SBF, Phygene Life Sciences Company Fujian, China) was used to test the scaffold's ability to interact with the body fluid after implantation.

2 Fabrication of bone scaffolds
First, HDDA and TPGDA were mixed at the mass ratio of 7 : 3. The mixture, together with Solsperse 41000 (4 wt% of ceramic powders) and Foamex N (0.1 wt% of ceramic powders), became the solvent after mixing evenly. Then, BCP (HA : β-TCP = 6 : 4) powders doped by different amount of T-ZnO w (0, 1, 2, and 3 wt% of ceramic powders, donated as Z0, Z1, Z2, Z3, respectively) were added into solvent, followed by stirring with a speed of 2800 r/min for 3 min in the vacuum planetary stirring defoaming machine (SIE-MIX80, Guangzhou Sienox Technology Co., Ltd., China). Solid loadings of all suspension were controlled at 40 vol%. Finally, TPO (2 wt% of total UV resin monomers) and MEHQ (0.2 wt% of total UV resin monomers) were added to prepare the final ceramic suspension for DLP. The TPMS-based double-layer scaffold model (diameter: 12.5 mm, height: 12.5 mm) with a 50% average porosity is shown in Fig. 2. The outer layer of the scaffold has a higher porosity (66%), which is beneficial for cells to grow in, while the inner layer is more compact (porosity 33%) to provide good mechanical properties. After being sliced by software with layer thickness set as 50 μm, models were printed by DLP printer (Autocera-M, Beijing Ten Dimensions Technology Co., Ltd., China) with a UV source of λ = 405 nm. Then the green bodies were cleaned in an ultrasonic bath to remove the uncured suspension. In the end, green bodies were debinded and sintered at 1200 ℃ as shown in Fig. 3.

3 Characterization
The rheological property of suspensions with different T-ZnO w contents was examined by Discovery DHR-2 rheometer (TA Instrument, New Castle, USA), with the shear rate ascending from 0.1 to 50 s −1 . The cure property was measured by single-layer printing [40]. Cure depth (C d ) was tested by micrometer, and for each test five independent thickness measurements were performed for averaging. Packing thickness was controlled at 800 μm.
To identify the influence of T-ZnO w to sinterability, the volume density and shrinkage of samples were tested using solid discs. Volume density was obtained by the Archimedes method. Shrinkage of porous scaffold was calculated according to Eq. (1): where l o is the size before sintering, and l s is the size after sintering. Three samples were tested for each composition. Scanning electron microscopy (SEM, Quanta650, FEI Company, USA) was used to observe the morphology of each sample, and X-ray diffraction (XRD, x'pert3 powder, PA Nalytical B.V., the Netherlands) was used to analyze the phase composition. Flexural strength was tested with a strip (5 mm × 4 mm × 40 mm) by three-point bending method, and compressive strength was tested with the porous double-layer scaffold shown in Fig. 2. The mechanical properties were all conducted on universal material testing machine (Zwick Z020, ZwickRoell, Germany).

4 Biological test
Apatite-forming test was conducted to access the in vitro bioactivity of the samples. The samples were immersed in the SBF (the ratio of SBF volume to sample weight is 200 mL:1 g) and vibrated at 37 ℃. After 1, 7, and 14 d, samples from each group were taken out. The microstructure of the surface was observed by SEM, and the functional group was tested by Fourier transform infrared spectroscopy (FTIR, Nicolet iS50R, Thermo Scientific, USA). The SBF solution was filtered, and then the concentration of Ca 2+ ion was tested by inductively coupled plasma optical emission spectrometer (ICP-OES, Prodigy Plus, Leeman Labs, USA) [41].
Considering high Zn 2+ concentration may be toxic for cells, and the critical concentration remains controversial [42,43], cytotoxicity was analyzed by the proliferation of cultured cells in extraction fluid of bioceramics [44,45]. Samples were washed with ethanol and sterilized at 121 ℃ for 20 min. MC-3T3 cells were cultured in α-MEM (Gibico, USA) supplemented with 10% fetal bovine serum (FBS, Gibico, USA), 1% antimicrobial of streptomycin and penicillin in a humidified atmosphere at 37 ℃ with 5% CO 2 . Cells at the third passage were used for the in vitro test. Cell counting kit (CCK-8, Boster, China) was used according to the manufacturer's protocol. To get the extraction fluid, every 200 mg bioceramic was immersed in 1 mL fluid and incubated at 37 ℃ for 24 h with a shaking speed of 120 r/min. Considering that the continuous flow of human blood would dilute the ion concentration, we prepared the extraction fluid diluted 4, 8, and 16 times at the same time. The cells were seeded at a density of 10 4 cells per well, and 100 μL extraction fluid was added to each well. After 1 and 3 d of cell culturing, 10 μL of CCK-8 reagent was added into the culturing medium 90 min before analysis at 37 ℃. The optical density (OD) values were read at 450 nm on an absorbance microplate reader (Bio-tek, USA).

1 Study on rheological and cure property of bioceramic suspension
The effect of T-ZnO w content on rheological property is shown in Fig. 4. Figure 4 suspensions to spread out [46]. With the increase of T-ZnO w content, the suspension viscosity decreases first and then increases. Z2 shows the lowest viscosity at about 0.45 Pas. Figure 4(b) exhibits the thixotropic loops. Area of the stress-shear rate loop, which can be calculated in Origin, reflects the thixotropy of suspension. Similar to the viscosity, the area of thixotropic loop also decreases first and then increases. Z2 has the smallest loop area, which means it is the easiest to recover after shearing, and help to reduce the formation of inner defect.
With the increase of T-ZnO w content, T-ZnO w can impede the aggregation of HA and β-TCP particles considering the unique tetrapod-like structure of the whiskers shown in Fig. 1(c), lowering the viscosity and the area of thixotropic loop. However, when the content of T-ZnO w keeps increasing, aggregation happens between whiskers [22], which reduce the dispersion effect, and increase the viscosity as well as the area of thixotropic loop. As shown in Fig. 5, when lnE i is 1.5, the cure depths are all larger than the printing layer thickness (50 μm). Though it can meet the energy required for monolayer curing, such cure is not thick enough to obtain a tight combination between layers, which makes it easy for samples to crack or even fall off during the printing process [33,47]. In the end, E i = 12 mJ/cm 2 (lnE i equals to 2.485) is chosen as the proper exposure energy, where the cure depth of suspension is about 90-110 μm, and Z3 has the smallest cure depth.

2 Performance of T-ZnO w doped BCP scaffolds
As shown in Fig. 6, double-layer porous scaffolds with high precision have been fabricated using the optimized printing parameters. From Figs. 6(a), 6(c), 6(e), and 6(g), it can be found that scaffolds are comprised of two parts, with high porosity outside and low porosity inside. Observing the enlarged figures (Figs. 6(b), 6(d), 6(f), and 6(h)), we can find that the surfaces of scaffolds are smooth on the whole except a few small pores on the surface caused by bubbles during the printing process. Z3 shows the worst accuracy compared with other samples considering that some of the pores outside are partly blocked. With the same exposure energy (dotted line in Fig. 5), Z3 has the lowest cure depth. In this way, more light is scattered and absorbed by surrounding suspension, resulting in excessive cure and decreased accuracy. Z0, Z1, and Z2 exhibit complete and interconnected pores with a pore size of about 500 μm, which is beneficial for the growth of bone tissue and blood vessel [48,49]. Figure 7 shows the phase composition of samples with different T-ZnO w contents sintering at 1200 ℃. As for Z0, only TCP and HA (JCPDS 09-0432) can be examined, and TCP exists in both α-TCP (JCPDS 09-0348) and β-TCP (JCPDS 09-0169) since the phase transition can take place on β-TCP at high temperature [50]. With the increase of T-ZnO w , HA shows no obvious differences, and remains to be the main phase, but obvious changes happen on TCP: Both β-TCP and α-TCP are weakened, and β-TCP almost disappears. Ca 19 Zn 2 (PO 4 ) 14 appears at the same time. These phenomena suggest that reaction  took place between T-ZnO w and β-TCP, causing the formation of new phase and inhibiting the phase transition [51]. Figure 8 shows the microscopic morphology of the sample surface and cross-section. It can be seen that there are still many pores in ceramics, and the pores left by T-ZnO w can be observed clearly. The crosssection of Z2 is rougher than others. In addition, a large number of needle-like structures with a length of about 10 μm can be seen on the surface and cross-section of Z1 (Figs. 8(c) and 8(d)) and the cross-section of Z2 ( Fig. 8(f)), indicating that new phase may form here.
The shrinkage, volume density, and mechanical strength of samples sintered at 1200 ℃ are shown in Fig. 9. Figure 9(a) shows the shrinkage rate both in transverse and longitudinal directions, from which we can find that longitudinal shrinkage is always larger than transverse shrinkage owing to the poor interlayer connection during printing. With the increase of T-ZnO w content, shrinkage and open density show no obvious difference. Figure 9(c) shows the flexural strength of the samples. With the content of T-ZnO w increases from 0 to 2 wt%, the flexural strength increases from 40.9 to 68.5 MPa. When the content of T-ZnO w keeps increasing to 3 wt%, the flexural strength decreases sharply to 49.4 MPa. This is because that four needles of T-ZnO w can disperse the stress to four directions effectively. Fracture or pulling out of whiskers can also absorb energy and inhibit the propagation of crack, and thus the chance of damage can be reduced. Then as Fig. 10 shows, when the content of T-ZnO w is 3 wt%, we can find that all fractures of Z3 show obvious step-like fracture, and the defect between layers is more obvious than that of Z2. It can infer that the low cure depth of Z3 cause delamination, and in turn decrease the flexural strength. However, despite the weak combination of printing, flexural strength of Z3 is still larger than that of Z0, which proves that the addition of T-ZnO w does help improve the flexural strength. The compressive strengths of Z0, Z1, Z2, and Z3 show no obvious difference ( Fig.  9(d)), and that of Z2 (18.31 MPa) is a little higher than others, which is consistent with the flexural strength.

3 In vitro bioactivity
SEM images of the samples after soaking for 1, 7, and 14 d are shown in Fig. 11. It can be seen that after soaking for 1 d, Z2 has the fastest mineralization rate. Surfaces of Z0 and Z2 have been covered by villous substances obviously and segmented into serious of "shell". By contrast, Z1 and Z3 have not been covered completely, but the surface of them has become rougher compared with that before soaking, and some wormlike particles appeared. By testing the ion concentration in SBF, we can find that the Ca 2+ concentration in Z2 is lower than others ( Fig. 12(a)), which means more Ca 2+ in SBF has deposited on the sample surface in the early stage, and formed the apatite mineralized layer. After soaking for 7 d, all the samples have been coated by villous substances intensively, forming a new spherical sediment layer with a diameter of about 4 μm, which is much larger than the original grain. The cracks in the sediment layer are caused by shrinkage during drying, proving that the sediment layer has a certain thickness.
After magnifying the surface ( Fig. 12(b)), it can be observed that the villous material is a layer of dense wormlike particles with a length of 200-300 nm. Energy spectrum scanning shows that a large number of C atoms can be found in these particles, indicating that HCO 3 ions in SBF solution may have been deposited on the surface. FTIR analysis exhibits the atom groups on the surface after soaking in SBF for 14 d (Fig.  12(c)). It shows strong absorption peaks of PO 4 3near 563 and 1000 cm −1 , and absorption peaks of CO 3 2functional group near 1430 cm −1 [52], which shows the existence of carbonated hydroxyapatite (CHA). CHA is similar to the inorganic components of natural bone in composition, and has large specific surface area. It can adsorb collagen, polysaccharide, and other substances, which is conducive to the adhesion and growth of osteoblast stem cells [53]. Therefore, the fast deposition rate of Z2 and well deposition of CHA on Z1 and Z3 proved that BCP ceramics containing T-ZnO w exhibit good in vitro bioactivity.
To test the cytotoxicity of the samples, cell proliferation test was conducted. The results are shown in Fig. 13. We can find that the OD values of 3 d are all obviously larger than those of 1 d, which means cells can proliferate well at different dilution times. Besides, there is no obviously difference between samples with   various T-ZnO w contents, indicating that current whisker contents are safe for cells.

Conclusions
In this work, T-ZnO w enhanced BCP scaffolds with TPMS-based double-layer porous structure were successfully fabricated by DLP technology. With low viscosity and optimized exposure energy (12 mJ/cm 2 ), scaffolds were all printed with high precision.
After samples being sintered at 1200 ℃, the new phase Ca 19 Zn 2 (PO 4 ) 14 forms by the reaction between T-ZnO w and β-TCP, and the phase transition to α-TCP is inhibited. With the increase of T-ZnO w , the flexural strength first increases and then decreases. Z2 has the highest flexural strength (68.5 MPa) because T-ZnO w could absorb energy and play the role of toughening.
Excessive T-ZnO w leads to low cure depth and poor combination between layers as well as more printing defects, which would cause the samples to break along layers during flexural test. In apatite forming test, Z2 shows the most Ca 2+ deposition and the fastest mineralization rate after soaking for 1 d. After soaking for 7 d, all samples mineralize completely. In in vitro test, the samples exhibit good biocompatibility since cells can proliferate well under different dilute times. These findings confirmed that BCP scaffolds doped by T-ZnO w can be well-printed by DLP. The addition of T-ZnO w can improve the mechanical property of composite scaffolds and keep good biological property, so that the composite scaffolds are hopeful to be used in personalized repair of bone defect.